Numerous factors contribute to the overall effectiveness of a given vaccine; these include specificity, magnitude and tempo of the response, efficiency of inducing memory, and avoidance of reactogenic effects. All of these concerns also apply to the design of adjuvants as a necessary component of virtually all vaccine approaches. This proposal focuses on the application of C3d as a natural molecular adjuvant with increased potency in promoting antigen-specific humoral responses in the absence of co-factors that promote non-specific immunity. Here we will investigate the suitability of C3d conjugation to the protective antigen (PA) of anthrax toxin as a candidate subunit vaccine approach to combat exposure to B. anthracis spores or purified exotoxins. C3d-PA fusion proteins and conjugates will be constructed, expressed and tested in mouse in vitro bioassay and in vivo challenge models. Extrapolation of these studies to the activation of PA-specific primary human B-cells will be examined by in vitro rechallenge of circulating memory B cells present in the peripheral blood of military personnel vaccinated during Gulf War I. Together, these studies should reveal the practical effectiveness and mode of action of C3dbound immunogens for potential use in vaccine approaches that require a robust and sustained antibody response. Pending completion of the proposed work outlined in this R21 application, a series of experiments involving anthrax challenge models would seem appropriate.